Epstein-Barr virus (EBV) causes or is associated with a wide range of diseases varying in severity from infectious mononucleosis to life-threatening lymphoproliferative diseases in immunocompromised patients. The consistent presence of EBV in particular tumor types, such as AIDS-associated CNS and other B-cell lymphomas, offers the potential for the development of highly specific, virus-targeted therapies. This proposal combines basic studies of a novel potential target, EBV BGLF4-encoded protein kinase (EBV PK), with applied studies, wherein the kinase will be tested as a "suicide" gene for treatment of EBV-positive and EBV negative tumors (in conjunction with ganciclovir treatment) or as a "therapeutic" gene for treatment of EBV driven lymphomas. In Aim 1, we study the role of EBV PK in viral infection by knocking out its expression using RNAi. We then analyze the course of viral lytic infection at the RNA and protein levels, starting with known EBV PK targets, such as viral DMA polymerase processivity factor, encoded by the EBV BMRF1 gene, and extending the research to newly discovered targets. In Aim 2, we investigate the phosphorylation of EBV EBNA2 protein, the master regulator of the EBV type 3 latency program characteristic of EBV lymphoproliferative diseases, and a newly discovered EBV PK target. Based on this finding, we also propose a novel potential therapeutic approach for treating EBV-driven lymphoproliferations, that is, by deregulation of type 3 EBV latency. In Aim 3, we develop ways to use EBV kinases, both PK and thymidine kinase (TK), to treat EBV-positive and EBV-negative malignancies. We study, first in cell culture and then in animal models of human disease, the efficacy of the kinases in converting ganciclovir to its cytotoxic form, and the ability of combinations of chemotherapeutic agents that trigger viral reactivation and ganciclovir to inhibit the growth of lymphomas containing wild type EBV or mutants lacking either kinase. Hence this research will reveal the role of EBV PK in viral infection and ultimately lead to new approaches for treatment of important EBV-related malignancies, based on new concepts rooted in fundamentals of the virology of EBV.